Precise control over axon branching is critical not only for the development of complex neuronal morphology and formation of neural circuits, but also for plasticity and regeneration after injury. Axon branching relies on tightly regulated dynamic cytoskeletal rearrangements, as well as trafficking and transport of cellular cargos such as membrane vesicles, proteins and organelles. How all these processes are orchestrated to ensure accurate neuronal branching patterns is still not well understood. The long-term goal of this project is to determine critical components of the axon transport machinery, and cargos such as guidance receptors, signaling mediators or cytoskeletal regulators that must coordinate to control axon branching. We established a model in which we can image dynamics of axon branching, neuronal cargo transport, and microtubule behavior in the intact zebrafish embryo. We previously found that a kinesin-cargo adaptor, Calsyntenin1 (Clstn1), is required for axon branching in sensory axons. Clstn1 is required for vesicular transport into developing axons and to branch points, and for organization of correct microtubule polarity during branching. We hypothesize that Clstn1 is a component of a coordinated cargo transport and microtubule organizing system that drives axon branching and controls where branches form. In Aim 1 we will combine CRISPR/Cas G0 phenotypic screening with rapid imaging using a unique light sheet microscope (Flamingo SPIM), and automated axon branch analysis to screen a set of candidate genes. Our goal is to identify genes required for axon branching and cargo transport, and genes that may interact with Clstn1. In Aim 2 we will use time-lapse imaging to determine effects of candidate gene mutation on motile axon behaviors, cargo transport and microtubule dynamics. Our goal is to further define molecular mechanism of action of genes important for branching. Elucidation of the molecular signals regulating sensory axon growth, branching, and protein trafficking is critical for understanding neurodegenerative disorders, neuropathic pain disorders and the conditions under which regeneration after axon injury can occur. Our experiments will uncover such mechanisms and thus may help to identify molecular targets for disease treatment.